The development of efficient and inexpensive genome sequencing methods has revolutionized the study of human bacterial pathogens and improved vaccine design. Unfortunately, the sequence of a single genome does not reflect how genetic variability drives pathogenesis within a bacterial species and also limits genome-wide screens for vaccine candidates or for antimicrobial targets. We have generated the genomic sequence of six strains representing the five major disease-causing serotypes of Streptococcus agalactiae, the main cause of neonatal infection in humans. Analysis of these genomes and those available in databases showed that the S. agalactiae species can be described by a pan-genome consisting of a core genome shared by all isolates, accounting for ≈80% of any single genome, plus a dispensable genome consisting of partially shared and strain-specific genes. Mathematical extrapolation of the data suggests that the gene reservoir available for inclusion in the S. agalactiae pan-genome is vast and that unique genes will continue to be identified even after sequencing hundreds of genomes.

https://www.pnas.org/content/pnas/102/39/13950.full.pdf

Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae: Implications for the microbial “pan-genome”

The traditional identification of bacteria on the basis of phenotypic characteristics is generally not as accurate as identification based on genotypic methods. Comparison of the bacterial 16S rRNA gene sequence has emerged as a preferred genetic technique. 16S rRNA gene sequence analysis can better identify poorly described, rarely isolated, or phenotypically aberrant strains, can be routinely used for identification of mycobacteria, and can lead to the recognition of novel pathogens and noncultured bacteria. Problems remain in that the sequences in some databases are not accurate, there is no consensus quantitative definition of genus or species based on 16S rRNA gene sequence data, the proliferation of species names based on minimal genetic and phenotypic differences raises communication difficulties, and microheterogeneity in 16S rRNA gene sequence within a species is common. Despite its accuracy, 16S rRNA gene sequence analysis lacks widespread use beyond the large and reference laboratories because of technical and cost considerations. Thus, a future challenge is to translate information from 16S rRNA gene sequencing into convenient biochemical testing schemes, making the accuracy of the genotypic identification available to the smaller and routine clinical microbiology laboratories.

Impact of 16S rRNA Gene Sequence Analysis for Identification of Bacteria on Clinical Microbiology and Infectious Diseases

Bacillus anthracis is the etiologic agent of anthrax, an acute fatal disease among mammals. It was thought to differ from Bacillus cereus, an opportunistic pathogen and cause of food poisoning, by the presence of plasmids pXO1 and pXO2, which encode the lethal toxin complex and the poly-γ-d-glutamic acid capsule, respectively. This work describes a non-B. anthracis isolate that possesses the anthrax toxin genes and is capable of causing a severe inhalation anthrax-like illness. Although initial phenotypic and 16S rRNA analysis identified this isolate as B. cereus, the rapid generation and analysis of a high-coverage draft genome sequence revealed the presence of a circular plasmid, named pBCXO1, with 99.6% similarity with the B. anthracis toxin-encoding plasmid, pXO1. Although homologues of the pXO2 encoded capsule genes were not found, a polysaccharide capsule cluster is encoded on a second, previously unidentified plasmid, pBC218. A/J mice challenged with B. cereus G9241 confirmed the virulence of this strain. These findings represent an example of how genomics could rapidly assist public health experts responding not only to clearly identified select agents but also to novel agents with similar pathogenic potentials. In this study, we combined a public health approach with genome analysis to provide insight into the correlation of phenotypic characteristics and their genetic basis.

https://www.pnas.org/content/pnas/101/22/8449.full.pdf

Identification of anthrax toxin genes in a Bacillus cereus associated with an illness resembling inhalation anthrax

Genomics has revolutionized every aspect of microbiology. Now, 13 years after the first bacterial genome was sequenced, it is important to pause and consider what has changed in microbiology research as a consequence of genomics. In this article, we review the evolving field of bacterial typing and the genomic technologies that enable comparative analysis of multiple genomes and the metagenomes of complex microbial environments, and address the implications of the genomic era for the future of microbiology.

/pdf/microbiology-in-the-post-genomic-era-1txoska6ng.pdf

Microbiology in the post-genomic era

SUMMARY A wide variety of microbial species are used in food and feed production. Some have a long history of apparent safe use, while others are less well understood and their use may represent a risk for consumers. Experience has shown that there is a need for a tool for setting priorities within the risk assessment of those microorganisms used in food/feed production referred to EFSA and consequently the subject of a formal assessment of safety. To meet this need a system was proposed for a pre-market safety assessment of selected groups of microorganisms leading to a “Qualified Presumption of Safety (QPS)”. In essence this proposed that a safety assessment of a defined taxonomic group (e.g. genus or group of related species) could be made based on four pillars (establishing identity, body of knowledge, possible pathogenicity and end use). If the taxonomic group did not raise safety concerns or, if safety concerns existed, but could be defined and excluded (the qualification) the grouping could be granted QPS status. Thereafter, any strain of microorganism the identity of which could be unambiguously established and assigned to a QPS group would be freed from the need for further safety assessment other than satisfying any qualifications specified. Microorganisms not considered suitable for QPS would remain subject to a full safety assessment. EFSA asked its Scientific Committee to consider whether this system could be used to harmonise approaches to the safety assessment of microorganisms across the various EFSA scientific panels. If so, the Committee was requested to develop a strategy for the introduction of an assessment system based on the QPS concept.

Introduction of a Qualified Presumption of Safety (QPS) approach for assessment of selected microorganisms referred to EFSA 1 Opinion of the Scientific Committee

In a bioterrorism event, a tool is needed to rapidly differentiate Bacillus anthracis from other closely related spore-forming Bacillus species. During the recent outbreak of bioterrorism-associated anthrax, we sequenced the 16S rRNA generom these species to evaluate the potential of 16S rRNA gene sequencing as a diagnostic tool. We found eight distinct 16S types among all 107 16S rRNA gene seqs fuences that differed from each other at 1 to 8 positions (0.06% to 0.5%). All 86 B. anthracis had an identical 16S gene sequence, designated type 6; 16S type 10 was seen in all B. thuringiensis strains; six other 16S types were found among the 10 B. cereus strains. This report describes the first demonstration of an exclusive association of a distinct 16S sequence with B. anthracis. Consequently, we were able to rapidly identify suspected isolates and to detect the B. anthracis 16S rRNA gene directly from culture-negative clinical specimens from seven patients with laboratory-confirmed anthrax.

/pdf/sequencing-of-16s-rrna-gene-a-rapid-tool-for-identification-3sn36z5xe8.pdf

Robin S. Weyant

Papers

Sequencing of 16S rRNA gene: a rapid tool for identification of Bacillus anthracis.